Characterization of transepithelial potential oscillations in the Drosophila Malpighian tubule.
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The Malpighian tubule of Drosophila melanogaster is a useful model system for studying the regulation of epithelial ion transport. In acutely isolated tubules, the transepithelial potential (TEP) undergoes large oscillations in amplitude with a period of approximately 30s. The TEP oscillations are diminished by reductions in the peritubular chloride concentration in a manner consistent with their being caused by fluctuations in chloride conductance. The oscillations are eliminated by pretreating tubules with the calcium chelator BAPTA-AM, although removal of peritubular calcium has no effect, suggesting that the oscillations are a result of either the release of calcium from intracellular stores or the entry of calcium from the tubule lumen. Transcripts encoding two calcium-release channels, the ryanodine receptor and the inositol trisphosphate receptor, are detectable in the tubule by reverse transcription-polymerase chain reaction. To identify the cell type responsible for the oscillations, tubules were treated with diuretic hormones known to alter calcium levels in each of the two cell types. Leucokinin-IV, which increases calcium levels in the stellate cells, suppressed the oscillations, whereas cardioacceleratory peptide 2b (CAP(2b)), which increases calcium levels in the principal cells, had no effect. These data are consistent with a model in which rhythmic changes in transepithelial chloride conductance, regulated by intracellular calcium levels in the stellate cells, cause the TEP oscillations. (+info)
The removal of sulphate by the excretory apparatus of the blowfly Calliphora vomitoria.
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The excretion of sulphate by the isolated Malpighian tubules of Calliphora vomitoria has been investigated. Contrary to expectation, it was found that the isolated tubules are freely permeable to sulphate. The rate of sulphate secretion is comparable to the rates of secretion of both phosphate and chloride. The excretion of sulphate by the intact fly has also been verified. (+info)
Characterization of the Drosophila melanogaster alkali-metal/proton exchanger (NHE) gene family.
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The NHE family of Na+/H+ exchangers is believed to play an essential role in animals, but may play an additional, specialised epithelial role in insects. The pharmacological sensitivity of the Drosophila melanogaster Malpighian tubule to a range of amiloride derivatives was shown to be consistent with an effect on an exchanger, rather than a Na+ channel. Consistent with this, no degenerin/epithelial Na+ channel (ENaC) genes could be detected in Malpighian tubules by reverse transcriptase/polymerase chain reaction (RT-PCR). Using a low-stringency homology searching, three members of the NHE family were identified in the genomic sequence of Drosophila melanogaster, although only two genes were represented as expressed sequence tags. All three genes (DmNHE1 at cytological position 21B1, DmNHE2 at 39B1 and DmNHE3 at 27A1) were found by RT-PCR to be widely expressed, and one (DmNHE2) was shown to have multiple transcripts. The putative translations of the three genes mark them as distantly related members of the family, inviting the possibility that they may serve distinct roles in insects. (+info)
Identification of a potent antidiuretic factor acting on beetle Malpighian tubules.
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Beetles, like other insects, depend on diuretic and antidiuretic hormones to control water balance. We have isolated, using head extracts from the beetle Tenebrio molitor, a peptide that strongly inhibits fluid secretion by the Malpighian tubules of this insect. This antidiuretic factor (ADF) appears to elicit its effect via cGMP as a second messenger but does not stimulate NO production. It has primary structure: Val-Val-Asn-Thr-Pro-Gly-His-Ala-Val-Ser-Tyr-His-Val-Tyr-OH. The ADF inhibits tubule secretion with high potency: the EC(50) is around 10 fM. It bears no significant resemblance to other biologically active neuropeptides. To our knowledge this is the only endogenous insect ADF acting on Malpighian tubules to be sequenced, and the first coleopteran (beetle) antidiuretic factor fully characterized to date. (+info)
Influx theory and size of potassium and rubidium pools in the midgut of Hyalophora cecropia.
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1. The midgut contains a 'pool' which must mix with tracer added to the blood-side, before the transported potassium reaches a steady specific activity. The size and mixing time of the pool can be deduced from the time-course of rate of appearance of tracer on the lumen-side, the tracer influx. The theory has been extended to cover cases where the pool-size and the transport rate vary with time. 2. By using 42K and 86Rb together, it is shown that the midgut treats potassium and rubidium differently, rubidium being transported nine-tenths as fast as potassium, when both are present in equal concentration. The influx pool for potassium is one-third larger than that for rubidium, but mixes four-fifths as fast with blood-side tracer. 3. The size of the potassium pool decreases with time, but that for rubidium does not, and the ratios of mixing times and transport rates are constant. 4. The implications of the results are discussed, both in terms of the accuracy of previous investigations and with respect to the probable intracellular location of part of the influx pool. (+info)
Emc, a negative HLH regulator with multiple functions in Drosophila development.
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Expression and functional analyses of Emc have demonstrated that it is a prototype for a protein required for multiple processes in development. Initially characterized as a negative regulator of sensory organ development, it was later found to regulate many other developmental processes and cell proliferation. Its ability to block the function of bHLH proteins by forming heterodimers, which are ineffective in DNA binding, accounts for the role of Emc in preventing the acquisition of several cell fates which are under the control of bHLH proteins. However, while maintaining this repressive molecular mechanism, emc also appears to act as a positive regulator of differentiation. (+info)
A genetic hierarchy establishes mitogenic signalling and mitotic competence in the renal tubules of Drosophila.
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Cell proliferation in the developing renal tubules of Drosophila is strikingly patterned, occurring in two phases to generate a consistent number of tubule cells. The later phase of cell division is promoted by EGF receptor signalling from a specialised subset of tubule cells, the tip cells, which express the protease Rhomboid and are thus able to secrete the EGF ligand, Spitz. We show that the response to EGF signalling, and in consequence cell division, is patterned by the specification of a second cell type in the tubules. These cells are primed to respond to EGF signalling by the transcription of two pathway effectors, PointedP2, which is phosphorylated on pathway activation, and Seven up. While expression of pointedP2 is induced by Wingless signalling, seven up is initiated in a subset of the PointedP2 cells through the activity of the proneural genes. We demonstrate that both signalling and responsive cells are set aside in each tubule primordium from a proneural gene-expressing cluster of cells, in a two-step process. First, a proneural cluster develops within the domain of Wingless-activated, pointedP2-expressing cells to initiate the co-expression of seven up. Second, lateral inhibition, mediated by the neurogenic genes, acts within this cluster of cells to segregate the tip cell precursor, in which proneural gene expression strengthens to initiate rhomboid expression. As a consequence, when the precursor cell divides, both daughters secrete Spitz and become signalling cells. Establishing domains of cells competent to transduce the EGF signal and divide ensures a rapid and reliable response to mitogenic signalling in the tubules and also imposes a limit on the extent of cell division, thus preventing tubule hyperplasia. (+info)
Antagonistic control of fluid secretion by the Malpighian tubules of Tenebrio molitor: effects of diuretic and antidiuretic peptides and their second messengers.
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Fluid secretion by insect Malpighian tubules is controlled by haemolymph-borne factors. The mealworm Tenebrio molitor provides the first known example of antagonistic interactions between endogenous neuropeptides acting on Malpighian tubules. The two corticotropin-releasing-factor (CRF)-related diuretic peptides previously isolated from Tenebrio molitor, Tenmo-DH(37) and Tenmo-DH(47), were found to stimulate Tenebrio molitor tubules in vitro in a dose-dependent manner with EC(50) values of 0.12 nmol l(-1) and 26 nmol l(-1) respectively. However, no synergistic or additive effect was observed when these two peptides were tested simultaneously. We then investigated antagonism between second messengers: dose-response curves were constructed for stimulation of Tenebrio molitor tubules by cyclic AMP and their inhibition by cyclic GMP. When both cyclic nucleotides were included in the bathing Ringer, the stimulatory effect of cyclic AMP was neutralised by cyclic GMP. Similarly, the stimulatory effect of Tenmo-DH(37) was reversed on addition of an antidiuretic peptide (Tenmo-ADF), which was recently isolated from Tenebrio molitor and acts via cyclic GMP. The cardioacceleratory peptide CAP(2b), originally isolated from Manduca sexta, also increases intracellular cyclic GMP levels and inhibited fluid secretion by Tenebrio molitor tubules, with an EC(50) value of 85 nmol l(-1). This inhibitory effect was reversed by Tenmo-DH(37). Endogenous diuretic and antidiuretic peptides, effective at low concentrations and acting via antagonistic second messengers, have the potential for fine control of secretion rates in the Malpighian tubules of Tenebrio molitor. (+info)